Project Summary Gene therapy is an emerging treatment strategy for epilepsy that promises to dampen activity in specific seizure related circuitry in order to prevent or lessen the intensity of seizures. Finding the appropriate target for delivery represents a significant challenge for preclinical seizure models. In order to optimize delivery parameters, multiple strategies, locations, and doses must be compared. We have developed a novel screening tool that uses optogenetic intensity-response curves to precisely determine thresholds for population discharge (aka. interictal spikes), the oPDT. Once this threshold is known, suprathreshold stimulus trains of varying length can be used to determine an after discharge threshold, a measure of seizure susceptibility. These two metrics can be collected in the same animals, compared, and tracked. Thresholds vary predictably with behavioral state (sleep/wake), but are stable over time allowing for multiple within subject experiments. A chronic multi-site array in hippocampus and connected structures allows for detection of network wide stimulus responses and also continuous monitoring of normal activity. We propose to test and optimize two promising gene therapy strategies using our optogenetic thresholding technique, in non-epileptic animals, in order to assess therapeutic potential. Kv1.1 overexpression in neurons reduces excitability by raising the functional threshold for activation and decreasing burst production. Kir4.1 overexpression in astrocytes improves their ability to absorb extracellular K+, preventing K+ build up and the resulting ictogenesis. In Aim 1, we will locate effective target areas and optimize the dose of Kv1.1 in order to balance efficacy with impairment of normal function. An AAV vector developed by our collaborator Edward Perez-Reyes will be used to deliver Kv1.1. Baseline activity, the oPDT, and the oADT will be tracked over time with multiple measurements taken before expression occurs (<2 weeks), while expression builds (2-6 weeks), and when expression levels stabilize (>6 weeks). Changes in these metrics over time will reveal important information about the circuit level effects of Kv1.1 overexpression and its viability as a treatment for epilepsy. In Aim 2, we will determine if Kir4.1 overexpression in astrocytes is sufficient to reduce seizure suseptability. An AAV vector specific for astrocytes (using the GFAP promoter), will be used to overexpress Kir4.1. Success in these experiments will help to identify potential targets for therapeutic intervention, assess the therapeutic window, and provide critical clues about the nature of population discharge and seizure generation.